Influence of Different β-Blockers on Platelet Aggregation in Patients With Coronary Artery Disease on Dual Antiplatelet Therapy

Original Manuscript

Influence of Different �-Blockers on PlateletAggregation in Patients With CoronaryArtery Disease on Dual Antiplatelet Therapy

Vladimir Ignjatovic, MD, PhD1, Sinisa Pavlovic, MD, PhD2,Vladimir Miloradovic, MD, PhD1, Nebojsa Andjelkovic, MD, PhD3,Goran Davidovic, MD, PhD1, Predrag Djurdjevic, MD, PhD3, Radojica Stolic, MD, PhD4,Violeta Iric-Cupic, MD, PhD1, Ivan Simic, MD, PhD1, Vesna D. Ignjatovic, MD, PhD5,Njegos Petrovic, MD1, Zorica Smiljanic, MD1, Vladimir Zdravkovic, MD, PhD1,Stefan Simovic, MD1, Danijela Jovanovic, MD, PhD3, and Jelena Nesic, MD6

AbstractIntroduction: The use of b-blockers in the treatment of patients with coronary heart disease is associated with a decrease in thefrequency of angina pectoris and mortality of patients. Due to the severity of the disease and previous cardiovascular inter-ventions, many patients with coronary artery disease (CAD) usee dual antiplatelet therapy to achieve greater inhibition of plateletaggregation. The influence of b-blockers on platelet aggregation in patients using antiplatelet therapy is not well understood.Objective: To examine the effect of different b-blockers on platelet aggregation in patients on dual antiplatelet therapy.Methodology: The study included 331 patients who were treated at the Department of Cardiology, Clinical Center Kragujevacduring 2011. Patients were divided into 4 groups depending on the type of b-blockers that were used (bisoprolol, nebivolol,metoprolol, and carvedilol). Platelet aggregation was measured using the multiplate analyzer and expressed through the value ofadenosine diphosphate (ADP) test (to assess the effect of clopidogrel), ASPI test (to assess the effect of acetyl salicylic acid), TRAPtest (to assess baseline platelet aggregation), and the ratio of ADP/TRAP and ASPI/TRAP ASPI/TRAP (ASPI - aranchidonic acidinduced aggregation, TRAP - thrombin receptor activating peptide) representing the degree of inhibition of platelet aggregationcompared to the basal value. In consideration were taken the representation of demographic, clinical characteristics, laboratoryparameters, and cardiovascular medications between the groups. Results: Patients who used nebivolol had a significantly lowervalue of the ratio of ADP/TRAP (0.39+ 0.30) compared to patients who used bisoprolol (0.48+ 0.26; P¼ .038), and trend towardthe lower values of ADP test (328.0+ 197.3 vs 403.7+ 213.2; P ¼ .059), while there was no statistically significant difference invalues of other laboratory parameters of platelet function between other groups. Conclusion: Patients with CAD on dualantiplatelet therapy who used nebivolol had significantly lower levels of residual ADP-induced platelet aggregation compared tobaseline than patients who used bisoprolol.

Keywordsb-adrenergic pathways, molecular biology, antithrombotic, acute myocardial infarction, angina, heart disease, ischemic, heartdisease

Introduction

The adenosine diphosphate (ADP) receptor antagonist, clopido-grel, in addition to acetyl salicylic acid (ASA) reduces the inci-dence of cardiovascular events in acute coronary syndrome andafter percutaneous coronary intervention with coronary stentimplantation.1,2

Measurements of platelet aggregability, after clopidogrel andASA administration, have shown heterogeneous response. Thevariation in response to antiplatelet therapy could be attributedto many factors: genetic (polymorphism of the P2Y12 gene,polymorphism of the P-450 CYP 3A4 gene, polymorphism or

1 Clinic for Cardiology, Clinical Center Kragujevac, Kragujevac, Serbia2 Pacemaker center, Clinical Center Serbia, Belgrade, Serbia3 Clinic for Hematology, Clinical Center Kragujevac, Kragujevac, Serbia4 Clinic for Nephrology and Urology, Clinical Center Kragujevac, Kragujevac,

Serbia5 Center for Nuclear Medicine, Clinical Center Kragujevac, Kragujevac, Serbia6 Center for Endocrinology, Clinical Center Kragujevac, Kragujevac, Serbia

Manuscript submitted: December 5, 2014; accepted: February 27, 2015.

Corresponding Author:

Stefan Simovic, Clinical Center Kragujevac, Zmaj Jovina 30, Kragujevac 34000,

Serbia.

Email: [email protected]

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mutation of cyclooxygenase [COX] 1, and overexpression ofCOX-2 gene), cellular (accelerated platelet turnover), or clinical(noncompliance, drug interaction, increased platelet reactivity,etc).3-7

Due to their antianginous effect, β-adrenergic blockers areoften prescribed to the patients with manifested coronary arterydisease (CAD).8 Several trials and 1 meta-analysis9 havereported antiaggregatory effect of some β-blockers, such as pro-pranolol,10 carvedilol,11 and nebivolol,12 but despite their exten-sive use in CAD, there isn’t much data on their antiaggregatorybenefits in patients already receiving dual antiplatelet therapy.Except for their common effect on β-adrenoceptor antagonism,β-blockers are a heterogeneous group of drugs that differ in theirβ1 selectivity, intrinsic activation, liposolubility, and in specificdrug properties such as induction of nitric oxide (NO) synthesis,α-receptor antagonism, membrane stabilization effect, and anti-oxidative effect which could potentially influence plateletfunction.

Aim

The aim of our study was to examine the influence of differenttypes of β-blockers (bisoprolol, carvedilol, nebivolol, and meto-prolol) on platelet aggregation induced by ADP, arachidonicacid, and thrombin receptor-activating protein in patients withCAD on dual antiplatelet therapy.

Methods

Our prospective, clinical study included 331 patients, of bothsexes, with no age restriction, hospitalized for treatment and/or diagnostics of coronary heart disease at Cardiology depart-ment, Clinical Center Kragujevac, Serbia, during 2011.

Study included only the patients who had documented CADexcept acute myocardial infarction, who were on antiplatelettherapy: ASA (100 mg daily) and clopidogrel (75 mg daily) forat least 1 week prior to study entry, and were using sameβ-blocker continuously for at least 1 month prior to study entry.

Patients who did not have continuity in the dual antiplatelettherapy (1 week prior to the study entry) or have stopped takingthe β-blockers, or had replaced it with other β-blockers 1 monthprior to the study entry, were excluded from our study. Patientswho concomitantly used other antiplatelet medications such asdipyridamole, abciximab, tirofiban, eptifibatib, and prasugrel,also were not included.

Laboratory Analysis and Data Collected

After inclusion of the patients in the study, during their hospi-talization, the blood samples were taken to determine the levelof platelet aggregation and other laboratory parameters such ascomplete blood count, hemostasis, lipidogram, glycaemia,C-reactive protein, blood urea nitrogen, serum creatinin, andenzymes (creatinine kinase [CK], CK-MB, lactate dehydro-genase, aspartate aminotransferase, and alanine transaminase).We also collected data about patients’ demographics, risk

factors for CAD, all medicaments patients were taking, indica-tions for hospitalization (urgent hospitalization—within1month since CADdiagnosis, elective—more than 1month sincediagnosis), history of all previous diseases, hospitalizations, andinterventions.

Platelet Function Monitoring

Platelet aggregability has been measured in heparinized wholeblood samples by the method of impedance aggregometry usingMultiplate analyzer (Dynabite, Munchen, Germany). The effectof antiplatelet therapy, ASA and clopidogrel, was assessed in2 ways. First, by direct measurements of platelet aggregabilityafter addition of agonist, ADP for the assessment of clopidogrelresponse (ADP test) and arachidonate for the assessment ofASA response (ASPI test), whereby higher values of these testsindicated a higher residual platelet aggregation and reducedantiplatelet effect of drugs. Second, by the ratio of ADP/TRAPor ASPI/TRAP (expressed as a percentage), which indicatespercentage of residual platelet activity, compared to the basalvalue. The TRAP test is used to assess the effect of inhibitorsof glycoprotein IIb/IIIa receptors on the platelet aggregability,and its value is not affected by treatment with ASA and the neg-ligibly by clopidogrel, which is the reason why this test is usedfor measuring basal platelet aggregability if patient didn’t takeglycoprotein IIb/IIIa antagonist.

Statistical Analysis

Data were analyzed by descriptive and analytical statistics.Depending on the obtained distributions, we used analysis ofvariance (ANOVA) or Kruskal-Wallis test for comparison ofgroups with different types of β-blockers in relation to the valueof ADP, ASPI, and TRAP tests. In order to reduce family-wiseerror rate in multiple comparisons, we used Bonferroni correc-tion, which is incorporated in post hoc analysis of ANOVA instatistical program SPSS. We also performed contrast tests forpost hoc testing of ANOVA results of interest.

We also used chi-square (χ2) test, t test, and Mann-WhitneyU test for analysis of influence of different demographic, clini-cal, and laboratory factors on the platelet aggregation testbetween the compared groups.

Results

Patients were divided into 4 groups depending on the typeof β-blockers that were used, whereas the largest group wasbisoprolol group and the smallest was metoprolol group.Patients’ demographics and clinical characteristics are shownin Table 1.

There were no significant differences in demographic charac-teristics between groups. All risk factors, except hypertension,were equally represented among groups. The highest percentageof patients who were treated for hypertension was in the group,which used bisoprolol, and the smallest in the metoprolol group.

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Between the groups, there was also no significant difference inthe useofother cardiovascular drugs, as shown inTable2.Nitrates,statins, ACE/AT inhibitors, ACE inhibitors/AT1 receptor blockerscalcium (Ca)-channel blockers, trimetazidine, and inhibitors ofproton pump were equally distributed among all groups.

Values of laboratory parameters among the groups are shownin Table 3, while the values of hemostasis parameters are shownin Table 4. Values of laboratory parameters and hemostasis para-meters, except for creatinine concentration, did not significantlydiffer between the groups that were compared.

Table 1. Patients’ Demographics and Clinical Characteristics.

b-Blocker Group Bisoprolol Nebivolol Carvedilol Metoprolol P Value

Number of patients (%) Total 157 (47%) 76 (23%) 54 (16%) 44 (13%)Age 62.32 + 9.48 63.08 + 8.98 66.11 + 9.0 63.56 + 9.08 .080a

Gender Male 63.7% 72.4% 61.1% 65.9% .517b

Female 36.3% 27.6% 38.9% 34.1%Indication Urgent 47.8% 36.8% 48.1% 52.3% .313b

Elective 52.2% 63.2% 51.9% 47.7%Previous events or intervention Angina pectoris 38.9% 32.9% 22.2% 40.9% .036b,c

Myocardial infarction 47.8% 46.1% 63.0% 54.5%PCI 10.2% 21.1% 11.1% 2.3%CABG 3.2% 0% 3.7% 2.3%

Hypertension Yes 93.0% 86.8% 94.4% 79.5% .030b,c

No 7.0% 13.2% 5.6% 20.5%HLP Hyperlipoproteinemia Yes 64.3% 63.2% 64.8% 70.5% .869b

No 35.7% 36.8% 35.2% 29.5%Smoking Yes 31.2% 26.3% 14.8% 36.4% .070b

No 68.8% 73.7% 85.2% 63.6%Heredity Yes 51.0% 46.1% 48.1% 59.1% .566b

No 49.0% 53.9% 51.9% 40.9%Diabetes mellitus Yes 28.7% 26.3% 35.2% 27.3% .719b

No 71.3% 73.7% 64.8% 72.7%

Abbreviations: ANOVA, analysis of variance; CABG, coronary artery bypass graft surgery; PCI, percutaneous coronary intervention; w2, chi-square.aANOVA test.bw2 test.cSignificant at P < .05.

Table 2. Use of Other Cardiovascular Drugs Between the Groups.

b-Blocker Group Bisoprolol Nebivolol Carvedilol Metoprolol P Value

Nitrates Yes 65.6% 57.9% 70.4% 72.7% .317a

No 34.4% 42.1% 29.6% 27.3%Statins No 20.4% 13.2% 11.1% 13.6% .302a

Atorvastatin 69.4% 71.1% 74.1% 61.4%Simvastatin 3.8% 7.9% 5.6% 13.6%Pravastatin 6.4% 7.9% 9.3% 11.4%

Proton pump inhibitors No 64.3% 76.3% 74.1% 75.0% .314a

Pantoprazole 30.6% 19.7% 25.9% 20.5%Omeprazole 5.1% 3.9% 0% 4.5%

ACE/AT inhibitors No 21.0% 30.3% 16.7% 22.7% .154a

Ramipril 45.9% 35.5% 35.2% 40.9%Quinapril 5.7% 7.9% 9.3% 11.4%Zofenopril 5.7% 2.6% 3.7% 0%Perindopril 5.1% 3.9% 3.7% 2.3%Fosinopril 9.6% 10.5% 18.5% 6.8%Enalapril 3.2% 2.6% 9.3% 15.9%Lisinopril 2.5% 3.9% 3.7% 0%Losartan 2.5% 3.9% 3.7% 0%

Trimetazidine Yes 15.3% 15.8% 20.4% 11.4% .674a

No 84.7% 84.2% 79.6% 88.6%Ca channel blockers Yes 17.8% 19.7% 24.1% 15.9% .720a

No 82.2% 80.3% 75.9% 84.1%

Abbreviations: Ca, calcium; w2, chi-square.aw2 test.

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Table 5 shows the mean, standard deviation, and median val-ues for ADP, ASPI, and TRAP test and the ratio of ADP/TRAPand ASPI/TRAP between the test groups.

The variables ADP, TRAP, and ADP/TRAP had normal dis-tribution, so we used ANOVA in order to analyze the differ-ences between 4 different β-blocker groups. For all variables,

ADP, TRAP, and ADP/TRAP, Levene test indicated homogene-ity of variances.

The ANOVA of the variable ADP indicated that differencesbetween the 4 compared groups were not significant (F3, 327 =2.502, P = .059; Figure 1A). Because of the borderline levelof significance, we have decided to make contrast analysis

Table 3. Values of Laboratory Parameters Between the Groups.

b-Blockers Bisoprolol Group Nebivolol Group Carvedilol Group Metoprolol Group P Value

AST Mean + SD 23.26 + 11.52 26.67 + 23.83 31.00 + 45.70 23.39 + 11.37 .610a

Median 21 21 21 21ALT Mean + SD 24.85 + 13.29 25.77 + 25.54 28.82 + 19.67 26.38 + 13.25 .563a

Median 21.5 21 24 23Cholesterol Mean + SD 4.84 + 1.39 4.70 + 1.37 4.37 + 1.40 5.17 + 1.54 .145a

Median 4.65 4.35 3.9 4.67TGL Mean + SD 1.56 + 0.86 1.48 + 0.98 1.49 + 0.99 1.9 + 1.03 .084a

Median 1.4 1.11 1.33 1.64LDL Mean + SD 3.10 + 1.23 2.77 + 0.99 2.87 + 1.28 3.19 + 1.27 .437a

Median 2.9 2.53 2.5 2.875HDL Mean + SD 0.95 + 0.34 1.12 + 0.64 0.95 + 0.36 1.07 + 0.30 .216a

Median 0.9 1.02 0.845 1Glucose Mean + SD 7.48 + 2.65 6.64 + 2.31 7.18 + 2.70 7.15 + 3.33 .424a

Median 6.0 5.8 6.3 5.7Urea Mean + SD 8.10 + 10.74 8.25 + 9.60 6.78 + 2.21 5.54 + 1.77 .100a

Median 5.6 6.2 6.6 5.6Creatinine Mean + SD 97.44 + 82.47 107.6 + 74.40 105.3 + 68.68 79.37 + 18.29 .028a,b

Median 82.0 89.0 92.5 72.5LDH Mean + SD 378.75 + 121.49 419.81 + 124.02 435.7 + 161.92 417.0 + 126.33 .198c

Median 371.0 387.0 429.5 440.0CK Mean + SD 153.8 + 391.66 133.82 + 183.17 243.7 + 678.28 143.2 + 290.08 .563c

Median 96.5 90.0 88.0 85.5C reactive protein Mean + SD 8.25 + 16.18 5.0 + 4.81 9.73 + 10.72 11.06 + 28.79 .184c

Median 5.0 5.0 5.25 5.0

Abbreviations: ANOVA, analysis of variance; AST, aspartate aminotransferase; ALT, alanine transaminase; CK, creatinine kinase; HDL, high-density lipoprotein;LDH, lactate dehydrogenase; LDL, low-density lipoprotein; SD, standard deviation; TGL, triglyceride; w2, chi-square.aw2 test.bSignificant at P < .05.cANOVA test.

Table 4. Values of Hemostasis Parameters Between the Groups.

b-Blockers Group Bisoprolol Nebivolol Carvedilol Metoprolol P Value

Leukocytes Mean + SD 7.49 + 1.97 7.0 + 1.66 7.47 + 2.20 6.99 + 2.19 .352a

Median 7.16 6.74 6.95 6.3Erythrocytes Mean + SD 4.44 + 0.54 4.48 + 0.40 4.39 + 0.50 4.56 + 0.39 .733b

Median 4.47 4.50 4.41 4.47Hemoglobin Mean + SD 134.77 + 17.32 137.14 + 13.18 132.77 + 16.54 138.88 + 14.24 .320a

Median 137.0 137.0 129.0 136.0Platelets Mean + SD 221.71 + 53.32 219.29 + 53.2 223.1 + 71.41 237.05 + 78.68 .594a

Median 218.0 207.0 222.0 222.0INR Mean + SD 1.01 + 0.15 1.03 + 0.13 1.09 + 0.45 1.01 + 0.11 .267b

Median 0.98 1.00 1.0 0.98aPTT Mean + SD 25.87 + 5.04 27.07 + 5.40 24.36 + 4.93 26.02 + 2.81 .176b

Median 25.0 26.0 25.0 25.0Fibrinogen Mean + SD 3.74 + 0.76 3.68 + 0.73 3.80 + 1.12 3.52 + 0.76 .594b

Median 3.64 3.60 3.62 3.40

Abbreviations: ANOVA, analysis of variance; aPPT, activated partial thromboplastin time; INR, International normalized ratio; SD, standard deviation; w2, chi-square.aANOVA test.bw2 test.

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Figure 1. A, Distribution of values of ADP test between the groups. B, Distribution of values of ASPI test between the groups. C, Distribution ofvaluesofADP/TRAPtestbetween the groups.D,Distributionof valuesofASPI/TRAPtestbetween the groups.ADP indicates adenosinediphosphate.

Table 5. Differences in Platelet Aggregability Values Among the Groups.

Test b-Blockers Bisoprolol Nebivolol Carvedilol Metoprolol P Value

ADP Mean + SD 403.7 + 213.2 328.0 + 197.3 365.9 + 199.6 389.1 + 169.8 .059a

Median 355 266 352.5 378.5ASPI Mean + SD 281.6 + 240.9 236.0 + 206.3 273.8 + 289.9 278.0 + 239.2

Median 178 164 181 190 .480b

TRAP Mean + SD 876.7 + 238.3 852.4 + 260.9 822.7 + 250.6 854.4 + 215.8 .575a

Median 892 854 797 860ADP/TRAP Mean + SD 0.48 + 0.26 0.39 + 0.30 0.46 + 0.23 0.48 + 0.20 .038a,c

Median 0.44 0.33 0.44 0.46Percent of reduction 48 39 46 48

ASPI/TRAP Mean + SD 0.30 + 0.24 0.29 + 0.26 0.30 + 0.24 0.33 + 0.26Median 0.22 0.22 0.23 0.25 .586b

Percent of reduction 22 22 23 25

Abbreviations: ADP, adenosine diphosphate; ANOVA, analysis of variance; SD, standard deviation.aANOVA test.bKruskal-Wallis test.cSignificant at P < .05.

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between the group with lowest ADP mean value, in contrast toall other groups combined, and, in second step, to compare itwith the group with highest ADP mean value. Contrast analysisindicated significantly lower mean in nebivolol group, than restof the 3 groups combined (t327 = 2121, P = .035), and signifi-cantly lower mean, compared to bisoprolol group (t327 = 2.679,P = .008), which could indicate that, despite nonsignificantfinding in ANOVA test, significant difference between nebivo-lol and bisoprolol group could be present.

The variable ASPI did not have normal distribution, so weperformed Kruskal-Wallis test, which indicated there were nosignificant differences between the 4 different β-blocker groups(Figure 1B). The ANOVA of the variable TRAP did not findsignificant differences between the 4 compared groups.

There was significant difference in the ANOVA test of thevariable ADP/TRAP between the compared groups for the 3conditions (F3,327 = 2.846, P = .038; Figure 1C). In order toidentify between which groups the difference was significant,and to decrease the probability of type 1 error in multiple com-parisons, we performed Bonferroni post hoc analysis, whichindicated that the mean value of the ADP/TRAP in nebivololgroup (0.39 ± 0.30) was significantly lower than the mean valueof the ADP/TRAP in bisoprolol group (0.48 ± 0.26). Post hoccontrast analysis indicated that nebivolol group had signifi-cantly lower mean of the ADP/TRAP value, than the rest of the3 groups combined (t327 = 2,612, P = .009), and also signifi-cantly lower compared to bisoprolol group only (t327 = 2.848,P = .005).

The variables ASPI/TRAP did not show normal distribution,so we performed Kruskal-Wallis test, which indicated therewere no significant differences between the 4 differentβ-blocker groups (Figure 1D).

Discussion

Numerous studies have demonstrated a modulation of plateletfunction and increased ADP-induced platelet aggregability bycirculating catecholamines.13,14 Some authors have foundincreased ADP and thromboxane-induced platelet aggregationin conditions where the catecholamine level is elevated, suchas in acute myocardial infarction15 during early morning hours16

or during physical17 or mental stress,18 although this was dis-puted by others.19,20

On the surface of the platelets prevail adrenoceptors of α2Asubtype and through their stimulation, epinephrine and norepi-nephrine potentiate the effects of other agonists and at higherconcentrations, initiate platelet responses. Human platelets pos-sess, in addition to α2 adrenoceptors, β2 adrenoceptors thatcould be of interest as potential target for β-blockers.21

Beres et al examined influence of adrenergic system on theplatelet function in patients who had angina pectoris and wereon dual antiplatelet therapy (ASA, clopidogrel). Patients whohad elevated adrenergic system activity (measured after admin-istration of low doses of adrenaline and selective α2A adrener-gic receptor inhibitor atipamezol) had higher levels of ADP andcollagen-induced platelet aggregation and weaker inhibitory

effect of thienopyridines measured with light transmissionaggregometry (LTA).22

We believe that in a state of elevated concentration of cate-cholamines use of β-blockers prevents excessive platelet activa-tion by catecholamines. The effect of β-blockers on plateletsshould not be limited only to the antagonistic effect of catecho-lamines. It is known that certain drugs from the group ofβ-blockers have membrane-stabilizing effect as well as othervarious additional effects: blockade of α adrenoceptors and sti-mulation of NO production from the endothelium.

Antiplatelet activity of nonselective β-blocker, propranolol,has been reported in several trials in the 1980s and 1990s.10,23

In the trial of Campbell et al,23 treatment of patients with hyper-tension having high-dose propranolol, significantly inhibitedthromboxane synthesis by platelets and platelet aggregationinduced by thrombin or arachidonic acid. In vitro study of pro-pranolol effect by Weksler et al10 on platelet aggregationshowed that propranolol inhibited ADP, epinephrine, collagen,and thrombin-induced platelet activation. This effect of the pro-pranolol is explained by membrane-stabilizing effect, affectingCa availability within the platelets and not by its β-blockingeffect.

Several smaller studies did not show antiplatelet effect of β 1selective blockers metoprolol and atenolol.24-26 The Scandina-vian group of authors24 examined the antiplatelet effect of meto-prolol on platelet aggregation induced by ADP in patients whohad myocardial infarction, 4 weeks before study entry. Theresearchers found no difference in ADP-induced platelet aggre-gation in 30 patients who took metoprolol compared to 33patients who were on placebo. Some authors demonstrated morepronounced antiaggregatory effect of propranolol in comparisonto metoprolol25 and atenolol.26

The antiaggregatory effect of nonselective β-blocker carvedi-lol, which is also α1 antagonist, has also been reported. Besidethis, α adrenoceptor antagonizing effect, this β-blocker hasmembrane-stabilizing effect. Petrikova and colleagues exam-ined the antiplatelet effect of carvedilol on platelet aggregationinduced by thrombin, epinephrine, and ADP measured by LTA,as well as the production of thromboxane B2, measured byradioimmunoassay. These results were then compared with theantiplatelet effect of atenolol and propranolol. Carvedilolexerted a pronounced antiplatelet effect in reducing platelet acti-vation and the formation of thromboxane B2 by propranolol,whereas atenolol did not show antiaggregational properties.10

Antiplatelet effect of carvedilol is explained by the effect of themacromolecules of the cell membrane (phospholipids, ion chan-nels, and enzymes).27

Zakirova and colleagues compared the effect of carvediloland metoprolol on platelet aggregation in patients after acuteST-segment elevation myocardial infarction using LTA. Thecarvedilol group consisted of 42 patients and the metoprololgroup of 44 patients. After 4 weeks follow-up, carvedilol grouphad lower ADP and collagen-induced platelet aggregation com-pared to the metoprolol group. Researchers also noted a smallermean platelet volume (MPV) in the carvedilol group, which isone of the predictors of outcome after myocardial infarction.28

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The most recent meta-analysis, which included 31 studiesabout antiplatelet effect of β-blockers, pointed out that clini-cally used doses of β-blockers reduce platelet aggregation, andthis effect was more pronounced in nonselective, lipophilicβ-blockers.9

Nebivolol is a novel, highly selective β-blocker with addi-tional vasodilatatory hemodynamic effects by acting on theL-arginine/NO pathway. In the study of Falciani et al12 nebivololinhibited ADP-induced platelet aggregation in greater extentthan propranolol and carvedilol.

Celik and colleagues compared antiplatelet effect of nebivo-lol and metoprolol in 72 patients measuring the MPV, concen-trations of P-selectin. After 6 months of follow-up, nebivololgroup had a significantly lower MPVand P-selectin blood level,compared to the metoprolol group.29

In examination of antiaggregatory effect of nebivolol andatenolol in Zucker-diabetic rats, authors have noted significantreduction in platelet aggregation and oxidative stress parametersin nebivolol group. Additionally, evaluation of expression ofplasminogen activator inhibitor and cellular molecules such asvascular cell adhesion molecule 1 and platelet endothelial celladhesion molecule 1 in the vascular wall of diabetic rats treatedwith nebivololol during 6 months showed favorable modifica-tion of these two prothrombotic markers compared to the con-trol group with atenolol.30

Group of Russian researchers examined effect of nebivololon the microcirculation, blood viscosity, spontaneous, andADP-induced platelet aggregation in 30 patients with moderatehypertension using laser-Doppler flowmetry. After 3 months oftreatment, patients had significantly reduced spontaneous andADP-induced platelet aggregation.31

Antiplatelet effect of nebivol can be explained by the influ-ence on the NO (L-arginine NO) system, especially on the activ-ity of endothelial form of NO synthase (eNOS).32,33 Thisantiaggregatory effect of nebivolol can be enhanced by addingthe substrate for eNOS, the amino acid L-arginine, and sup-pressed by adding of eNOS inhibitor (L-NAME).33

Reduced production of NO is one of the main causes ofendothelial dysfunction, which is closely associated with devel-opment of atherosclerosis. Stimulation of β adrenergic receptorsstimulates endothelial NO production, under the influence ofeNOS, but paradoxically excessive stimulation of this mechan-ism inhibits the synthesis of NO by creation of excessive reac-tive oxygen species (ROS, such as superoxide anion).34

Excessive activation of the sympathetic system and increasedconcentrations of catecholamines are one of the main factors forthe development of endothelial dysfunction due to blockade ofNO, increased production superoxide anion, and synthesis ofinflammatory mediators.35,36

In addition, NO plays a key role in maintenance of the nor-mal function of the vascular endothelium. It reduces adhesionand aggregation of the platelets, inhibits thrombus formation,adhesion of monocytes to the vascular endothelium, and smoothmuscle cell proliferation.37 NO is also part of the protectivemechanism by limiting inflammation of the endothelium and theproduction of tissue factor.38

Antioxidant properties of nebivolol have been demonstrated inthe laboratory and clinical trials. Through interaction with ROS,39

nebivolol reduced concentration of peroxynitrite.40 In two studiesauthors noted reduced NO inactivation by ROS in the group ofpatients with nebivolol, compared to the control group in patientswith hypertension.41,42 In experimental models of hypertension,hypertension of angiotensin II-treated rats, spontaneously hyper-tensive rats, and rats with transgenic overexpression of renin,nebivolol inhibited nicotinamide adenine dinucleotide phosphateoxidase and reduced oxidative stress.43-45

Induction of NO synthesis and neutralizing ROS together withinhibiton of β adrenergic receptors synergistically contribute tothe preservation of endothelial function after administration ofnebivolol and may explain more pronounced atiaggregatoryeffect of nebivolol in comparison to bisoprolol in our trial.

Process of platelet activation aggregation is influenced bynumerous factors. During data collection and analysis, we con-sidered all demographic, clinical, and laboratory factors thatcould affect aggregation test, but did not find significant differ-ence in their distribution between the groups, with exception ofhypertension, which was lowest in metoprolol (79.5%) andhighest in bisoprolol and carvedilol group (93% and 94.4%,respectively). Values of laboratory parameters including hemos-tasis parameters and inflammation markers did not significantlydiffer between the compared groups. In our study, we observeddifferent levels of reduction in ADP-induced platelet aggrega-tion compared to baseline values (ADP/TRAP) and trendtoward lower ADP-induced platelet aggregation between thegroups of patients on dual antiplatelet therapy who used nebivo-lol and bisoprolol, which cannot be attributed to other factorsthan individual properties of those different β-blockers.

Metoprolol and carvedilol group had a similar level of ADPand arachidonate-induced platelet aggregation, as bisoprololgroup, which indicate their similar effect on the platelet aggre-gation. Despite this, we didn’t find significant difference inADP test value nor in the relationship ADP/TRAP test hadbetween metoprolol and carvedilol groups, in comparison withnebivolol group. This could be explained by the smaller numberof patients in those 2 groups (54 patient in carvedilol, 44 inmetoprolol group) in comparison with bisoprolol group (157patient), so observed differences in the values of aggregationtests could not manifest.

We acknowledge that smaller number of patients in the meto-prolol and carvedilol groups as well as unequal group sizesamong the groups could be one of the limitations of the of study.We can also assume that some of the observed differences in thevalues of aggregation test (especially regarding ADP test) couldnot manifest due to the smaller number of patients included inour study, so further research with larger number of patients isneeded.

Conclusion

In patients with verified CAD on dual antiplatelet therapywe found significantly lower ADP-induced residual plateletaggregation compared to baseline values in the group of patients

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who used the nebivolol compared to the group of patients whoused the bisoprolol.

There was no significant difference in neither ADP, nor ara-chidonic acid-induced platelet aggregation between the groupsof patients who used different types of β-blockers (bisoprolol,nebivolol, carvedilol, and metoprolol). We have noticed a trendtoward slower ADP-induced platelet aggregation in a group ofpatients who used the nebivolol in comparison to group whoused bisoprolol. Therefore, we may conclude that nebivololcould be a better choice of β-blocker therapy, in patients withCAD, and on dual antiplatelet therapy, compared with otherβ-blockers (bisoprolol, carvedilol, and metoprolol).

Author Contribution

V. Ignjatovic, S. Pavlovic, and D. V. Ignjatovic contributed to concep-tion or design; acquisition, analysis, or interpretation; drafted the arti-cle; critically revised the article; gave final approval; and agree to beaccountable for all aspects of work ensuring integrity and accuracy.V. Miloradovic, G. Davidovic, P. Djurdjevic, and V. Iric-Cupic contrib-uted to conception or design; acquisition, analysis, or interpretation; andgave final approval. N. Andjelkovic, R. Stolic, N. Petrovic, Z. Smiljanic,D. Jovanovic, and J. Nesic contributed to acquisition, analysis, orinterpretation and gave final approval. I. Simic, V. Zdravkovic, andS. Simovic critically revised the article and gave final approval.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect tothe research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship,and/or publication of this article.

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